The long term goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the human erythrocyte ankyrin gene, ANK1. Erythrocyte ankyrin, an important component of the erythrocyte membrane skeleton, is the prototype of a family of homologous proteins that serve as linker/adaptor molecules between integral membrane proteins and the spectrin-actin based membrane skeleton. The studies in this proposal include the identification of ankyrin mutations n patients with hereditary spherocytosis (HS), a common inherited hemolytic anemia, and characterization of the structural and/or functional significance of these abnormalities. Another aim of this proposal is the identification and analysis of cis-acting sequences and trans-acting factors that regulate expression of the erythrocyte ankyrin gene. These results will be applied to the genetic study of HS patients who have been found to have mutations in the erythroid ankyrin gene promoter. A third aim of this proposal is the characterization of the neural isoform(s) of the ANK1 gene, identification of cis-acting sequences and trans-acting factors that regulate neural-specific expression of the ANK1 gene, and immunolocalization of these neural isoform(s). The final goal of this proposal is the characterization of the developmental and tissue-stage specific pattern of expression directed by key regulatory elements of the ANK1 gene in vivo. The general methodology to be utilized in this research includes: study of genomic DNA from individuals with ankyrin- related HS using two PCR-based mutation detection methods, single stranded conformational polymorphism analysis, and enzyme mismatch cleavage, followed by nucleotide sequence of PCR-amplified DNA; analysis of function of mutant recombinant peptides synthesized in vitro; cloning and structural analysis of the cDNA and genomic DNA segments of the ankyrin gene relevant to its expression and regulation by the use of recombinant DNA technology; study of cis-acting sequences by gene manipulation followed by gene transfer/expression studies in tissue culture cells; studies of trans-acting factors by electrophoretic mobility shift assays, DNAse-I footprinting, methylation interference techniques and site-directed mutagenesis followed by in vitro and in vivo analyses, and guanine-adenine ligation-mediated polymerase chain reaction (GA- LMPCR) dimethyl sulfate in vivo footprinting; developmental and tissue- specific studies of the regulatory sequences of the ANK1 gene promoters in transgenic mice using reporter gene assays. These studies will provide important insights into our understanding of the role of ankyrin in normal and disease states.